Compositions and methods for modulating the immune system

ABSTRACT

A novel class of embryo derived peptides are described (Preimplantation factor) that were generated synthetically and were tested on peripheral blood immune cells and shown to block activated but not basal immunity, inhibiting cell proliferation and creating a T H 2 type cytokine bias, in addition PIF enhance endometrial receptivity by increasing adhesion molecules expression. PIF biological activity appears to be exerted by specific binding to inducible receptors present on the several white cell lineages. PIF peptides, which are immune modulators therefore may have diagnostic and non toxic therapeutic applications in improving fertility, reducing pregnancy loss as well may be useful when administered for the treatment of autoimmune diseases and for prevention xenotransplants rejection.

This application is a continuation of U.S. application Ser. No.12/786,290 entitled “Compositions and Methods for Modulating the ImmuneSystem” filed May 24, 2010, which is a continuation of U.S. applicationSer. No. 11/381,818 entitled “Compositions and Methods for Modulatingthe Immune System” filed May 5, 2006, now U.S. Pat. No. 7,723,290,issued May 25, 2010, which claims priority from U.S. ProvisionalApplication No. 60/765,400 entitled “Characterization of PreimplantationFactor 1 (PIF-11): An Embryo-Derived Peptide with Immune ModulatoryProperties” filed Feb. 3, 2006, U.S. Provisional Application No.60/765,393 entitled “PIF-1: An Embryo-Derived Peptide PreventsAutoimmune Disease Development in Preclinical Trials” filed Feb. 3,2006, and U.S. Provisional Application No. 60/765,398 entitled “GVHDTherapy In Cancer Patients Using PIF (Preimplantation Factor), A NonToxic Embryo-Derived Immunemodulatory Peptide” filed Feb. 3, 2006, andU.S. Provisional Application No. 60/746,511 entitled “PIF-1 InducedEffects on PBMC Genome Alone and Following Exposure to CD3MAB/CD28MAB”filed May 5, 2006. U.S. application Ser. No. 11/381,818 is also acontinuation-in-part of U.S. application Ser. No. 10/971,760 entitled“Methods of Administering PIF Agonist Peptides and Uses Thereof” filedOct. 22, 2004, now U.S. Pat. No. 8,222,211 issued Jul. 17, 2012, andU.S. application Ser. No. 11/110,990 entitled “PIF Tetrapeptides” filedApr. 20, 2005, now U.S. Pat. No. 7,723,289 issued May 25, 2010. Allaforementioned applications are herein incorporated by reference intheir entireties.

BACKGROUND

Mammalian pregnancy is a unique physiological event in which thematernal immune system interacts with the fetus in a very efficientmanner, beneficial for both parties. Pregnancy is an immune paradox,displaying no graft vs. host or host vs. graft effect. The factorsinvolved in this phenomenon are not yet fully elucidated although theyhave been extensively studied. The novel embryo-derived factor,preimplantation factor (PIF-1), may cause immune tolerance of pregnancyby creating maternal recognition of pregnancy shortly afterfertilization. Synthetic PIF-1 replicated the native peptide's effectand exerted potent immune modulatory effects on activated PBMCproliferation and cytokine secretion, acting through novel sites on PBMCand having an effect which is distinct from known immune-suppressivedrugs.

There is evidence that several autoimmune diseases, including multiplesclerosis and rheumatoid arthritis, undergo remission during pregnancy,supporting the view that there are unique protective mechanismsoperative during that time period. This is particularly remarkablebecause the host/mother is simultaneously being exposed to a semi- ortotal allograft (donor embryo) without adverse immune effects.

Allogeneic bone marrow transplantation (BMT) is a well-establishedtreatment for malignant and non-malignant hematological diseases, and isperformed in tens of thousands of patients each year. Mature donor Tcells within the stem cell graft are the main mediators of thebeneficial immune effects, but they are also responsible for theinduction of graft-versus-host disease (GVHD), the major cause ofmorbidity and mortality in BMT patients. GVHD occurs when transplanteddonor-derived T cells recognize proteins expressed by recipientantigen-presenting cells. Consequently, this recognition induces donorT-cell activation, proliferation, and differentiation, leading to acellular and inflammatory attack on recipient target tissues. Acute orchronic GVHD occurs within a 100-day period post-BMT that leads todermatitis, enteritis, and hepatitis. The treatment of GVHD continues tobe a challenge. To eliminate undesirable host-derived hematopoieticelements before BMT, patients have traditionally been treated withmyeloablative conditioning regimens involving high-dose chemotherapy andtotal-body radiation. Up until now, standard GVHD prophylaxis andtherapy uses immune suppressive drugs (steroids and Cyclosporin A), thatplace patients in danger of opportunistic infections and tumor relapse.Numerous agents have been evaluated for GVHD, unfortunately with pooroutcome. Ideally, prophylaxis of BMT patients by immune modulation wouldallow transplant acceptance, while maintaining the ability to protectagainst pathogens or cancer.

Type 1 (insulin-dependent) diabetes (TIDM) is caused by autoimmunedestruction of the insulin-producing pancreatic beta cells. TIDMetiology is multifactorial, complex, and involves a combination ofgenetic, environmental, and immunological influences. TIDM is aprogressive, asymptomatic decline in beta cell function untilhyperglycemia develops. Near total beta-cell destruction may not beuniversal, and therefore therapeutic measures that stop destruction andperhaps lead to organ recovery could bring to major advances in TIDMmanagement. TIDM prevention is currently suboptimal, and most currenttherapies aim at controlling glucose levels using insulin, or (rarely)by islet transplants. There are also attempts to initiate immunetherapies using (anti-CD3 antibodies, and anti-thymocyte globulin) whichaim to block the autoimmune cascade when combined withrepair/regeneration of beta cells (e.g., glulisine,glucagon-like-peptide-1 (GLP-1), extendin-4), and Dia-Pep277 withlimited success.

Multiple sclerosis (MS) is a progressive debilitating autoimmune diseaseof the central nervous system that has a complex etiology where geneticpredisposition may be coupled with early childhood viral exposure.Consequently, there is a gradual destruction of the myelin sheath thatcauses motor, autonomic, sensory dysfunction that may lead to paralysis.Current therapies are based on limiting the damage by using steroids andinterferon, Copaxone and monoclonal antibodies, with limited success. Anoptimal therapy would reverse the neural damage by blocking theautoimmune cascade while allowing for myelin sheath repair. Theexperimental autoimmune encephalitis (EAE) model is widely usedcurrently to examine experimental treatments for MS.

Ulcerative colitis (UC) and Crohn's disease (CD), the primaryconstituents of inflammatory bowel disease (IBD), are precipitated by acomplex interaction of environmental, genetic, and immunoregulatoryfactors. Higher rates of IBD are seen in northern, industrializedcountries. IBD's are chronic inflammatory disorders of thegastrointestinal tract. Although the etiology is incompletelyunderstood, initiation and aggravation of the inflammatory process seemto be due to a massive local mucosal immune response. Cytokine-mediatedimpairment of viability and metabolic function of epithelial cells hasbeen suggested as a possible early pathogenic event in the developmentof inflammatory bowel disease (IBD). Among several currently usedtherapies are azulphidine, steroids and in more serious casesAzathioprine, 6-mercaptopurine and methotrexate are appropriate. Whensteroids fail, cyclosporine A may utilized. IBD's involve both local andsystemic alteration of the immune system. In recent years severalstudies were carried out using peripheral immune cells as well colonicbiopsies to examine the direct effect of possible therapeutic agents onthe condition. Data indicates that the milieu of peripheral PBMC isaltered and agents that were found to be disease modifiers by in situtesting were considered suitable for clinical application.

It has been observed that PIF has immune modulatory properties and suchpeptides are useful in the prevention and/or treatment of variousimmune-mediated diseases, including, but not limited to, autoimmunedisorders. Compositions and methods for treating and/or preventingimmune-mediated disorders are provided herein.

SUMMARY

Embodiments of the present invention provide compounds havingimmune-modulating and/or anti-inflammatory activity, wherein compoundsof the invention include peptides and peptidomimetics. The inventionfurther provides methods of using immune-modulating and/oranti-inflammatory compounds of the invention.

Embodiments of the present invention relate to biological effectsinduced in vitro and/or in vivo by pre-implantation factor, (PIF),peptides, peptidomimetics, and compounds derived from pre-implantationembryos that harbors in part, is identical to, or is homologous to theamino acid sequence of PIF peptides or to the scrambled amino acidsequence of PIF peptides. The invention also relates to the developmentof antibodies to quantitatively detect PIFs peptides in biologicalfluids.

The present invention also relates to the use of PIF antibodies forimmunocytochemical and Western blot to identify PIF related proteins inpregnant tissues, fetus and placenta. This allows identifying pregnancypathologies like premature labor and growth restriction as non-limitingexamples. The PIF antibodies used as affinity column can identifyassociated functional proteins in pregnant tissues as seen byidentification of 10 distinct proteins in the term placenta, several ofthem novel for that tissue. In such embodiment, other biomarkers can beidentified that may be modified by pregnancy disorders. Identificationof these proteins allows the examination of the genes that areassociated with these proteins highly relevant for blastocystdevelopment. These proteins using mass spectrometry or antibodies canalso aid together with PIF peptides to determine embryo viabilityfollowing in vitro fertilization thereby increasing the chances forpregnancy following transfer.

Further embodiments of the present invention provide methods fortreating a disease characterized by an immune disorder or inflammatoryresponse by administering an amount of a PIF peptide or peptidomimeticsufficient to treat, inhibit, or ameliorate the disease. Such compoundsare useful for treating diseases characterized by an immune disorder orinflammatory response diseases, e.g., inflammation, arthritis,auto-immune diseases, collagen diseases, or allergy. For example, thesecompounds can be used to treat subjects, including mammals such ashumans, having or at risk of having inflammation, arthritis, auto-immunediseases, collagen diseases, or allergy.

Embodiments of the present invention relate to biological effectsinduced in vitro and/or in vivo by pre-implantation factor (PIF)peptides, peptidomimetics, and compounds derived from pre-implantationembryos that harbors in part, is identical to, or is homologous to theamino acid sequence of PIF peptides or to the scrambled amino acidsequence of PIF peptides. In particular, the present invention relatesto use of PIF peptides or peptidomimetics to effect changes on theimmune system of a patient. More specifically, the addition of PIFpeptides creates specific changes both in cellular immunity, as well asin a patient's secreted cytokine profile.

DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1. PIF prevents GVHD development following high burden BMT. Thenumber of GVHD+ and GVHD− mice at three and five weeks after BMT wereevaluated. The differences between control and both 0.1 and 1 mg/kg/dayPIF administered for two weeks PIF group and tested one week later weresignificant. Also, the 1 mg/kg/day PIF-treated group using an Alzet®pump at five weeks after BMT provided significant protection, χ²:P≦0.001, P≦0.01 and P≦0.05, respectively.

FIG. 2. PIF-treated mice with a high BMT burden that develop GVHD have alower score at 30 days post transplant. At 30 days post-BMT, thedifference between the control and the 0.1 and 1 mg/kg/day treated PIFgroups using an Alzet® pump, are significant. t-test P≦0.04 and P≦0.04.At 50 days, the effect was not significant.

FIG. 3. Short-term PIF administration to mice with high-burden BMT isassociated with long term survival. PIF 1-5 mg/kg/day for two weeks wasadministered using an Alzet® pump and the effect on long-term survivalwas compared to control. In the lower-dose PIF-treated group, 7/9 micesurvived vs. control 2/10, χ², P<0.01. The survival of the higher-dosetreated group was slightly lower, 5/9.

FIG. 4. PIF treatment prevents diabetes development in NOD male mice,adoptive transfer model. Male mice were injected IV with 250M spleencells derived from diabetic female NOD mice. PIF 0.83-2.73 mg/kg/day wasadministered using Alzet® pump for 28 days. This was followed by a40-day observation period. In low-dose PIF group, no mice developed DM,while in the high-dose group, one mouse became diabetic vs. 6/7 incontrols, P<0.001.

FIG. 5A. sPIF-115 (SEQ ID NO: 13) ELISA standard curve PIF antibodydetects low sPIF levels (pg). Polyclonal antibodies AbPIF-115 weregenerated against sPIF-115 (SEQ ID NO: 13) in rabbits (Covance Inc.).High titers 50% at 1:50,000 were achieved. Serial dilutions of syntheticsPIF-115 (SEQ ID NO: 13) were plated, blocked and then washed off. PIF-1antibody (1:5000) was added incubated and washed off. Goat anti-rabbitantibody was added, incubated and washed off. Reaction was stopped bySDS and counted in plate reader (Biosynthesis Inc, G Vandydriff). Theantibody affinity was also confirmed by using a competition analysisbetween biotin labeled and unlabeled sPIF-115 (SEQ ID NO: 13) (data notshown). Also, when scrPIF-1 (SEQ ID NO: 5) was tested in the assay theantibody did not recognize it attesting to the high specificity of theantibody that was generated. Similar dose dependent results in the ELISAwere obtained with affinity purified PIF-2 and PIF-3 antibodies(dilutions of the antibody up to 25,600) with linearity to the 30 μM ofthe peptide.

FIG. 5B shows ELISA profile of high affinity PIF-1 IgY antibodies(sandwich assay). Chicken were injected with KLH bound PIF-1 and theeggs were collected and affinity purified on a PIF column.

FIG. 6 shows ELISA profile of nPIF-115 and scrPIF-115 using Biotinlabeled versus unlabeled peptide where the antibody captures the peptidein the unknown samples and compares it to standards (see FIG. 7).

FIG. 7 depicts an example of a PIF-based diagnostic of the presentinvention. Four clones of monoclonal antibodies to PIF-115 weredeveloped as well in mice and ascites fluid was generated with highaffinity antibodies as hybridomas with sustained MAb production.

FIG. 8. nPIF-115 (SEQ ID NO: 1) is present in the ovine placenta, asdemonstrated by immunocytochemistry methods. AbPIF-115 was exposed toplacental tissue derived from a midtrimester ovine fetus. Compared tothe non-specific staining by rabbit IgG, the PIF has shown intensestaining of the fetal portion of the placenta.

FIG. 9. Human term placental extracts were prepared (Dr Jerry Feitelson,GenWay, Inc) and were exposed to PIF antibodies using Western blotanalysis, PIF like molecules were stained and the bands obtained werecompared to a serial molecular weight standards run in parallel. Resultsshowed that a number of PIF-1 related proteins are present at the 15-40kDa range PIF-3 had a lower intensity with and was associated withdifferent molecular weight bands. Finally, PIF-2 expression was minimal.This supports the notion that the human placenta may have precursorproteins from which by cleavage PIF peptides are produced. This alsosupports that view that PIF like molecules are present throughoutpregnancy. Finally, it documents that in terms of intensity ofexpression in human by far PIF-1 is the most relevant at term.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of the present invention, the preferred methods, devices,and materials are now described. All publications mentioned herein areincorporated by reference. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “administering”, when used in conjunction with PIF, can include,but is not limited to, providing PIF peptide into or onto the targettissue; providing PIF peptide systemically to a patient by, e.g.,intravenous injection whereby the therapeutic reaches the target;providing PIF peptide in the form of the encoding sequence thereof tothe target (e.g., by so-called gene-therapy techniques). “Administering”a composition may be accomplished by parenteral, oral or topicaladministration.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.In a preferred embodiment, the therapeutic composition is notimmunogenic when administered to a subject for therapeutic purposes.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a subject. In part, embodiments of the present invention are directedto treating, ameloriating, preventing or improving inflammation and/oran immune-mediate disorder, including auto-immune diseases.

A “therapeutically effective amount” or “effective amount” of acomposition is a predetermined amount calculated to achieve the desiredeffect, i.e., to effectively inhibit or reduce inflammation and/or animmune-mediated disease. Effective amounts of compounds of the presentinvention can objectively or subjectively reduce or decrease theseverity or frequency of symptoms associated with inflammation and/orimmune-mediated disorders. The specific dose of a compound administeredaccording to this invention to obtain therapeutic and/or prophylacticeffects will, of course, be determined by the particular circumstancessurrounding the case, including, for example, the compound administered,the route of administration, and the condition being treated. Thecompounds are effective over a wide dosage range and, for example,dosages per day will normally fall within the range of from about 0.01mg/kg to about 10 mg/kg, more preferably about 0.1 mg/kg to about 1mg/kg. However, it will be understood that the effective amountadministered will be determined by the physician in the light of therelevant circumstances including the condition to be treated, the choiceof compound to be administered, and the chosen route of administration,and therefore the above dosage ranges are not intended to limit thescope of the invention in any way. A therapeutically effective amount ofcompound of this invention is typically an amount such that when it isadministered in a physiologically tolerable excipient composition, it issufficient to achieve an effective systemic concentration or localconcentration in the tissue.

The terms “treat,” “treated,” or “treating” as used herein refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or to obtain beneficial ordesired clinical results. For the purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationof symptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

“Disease” or “disorder” refers to an impairment of the normal functionof an organism. As used herein, a disease may be characterized by, e.g.,an immune disorder or an inflammatory response or a combination of theseconditions.

“Immune-modulating” refers to the ability of a compound of the presentinvention to alter (modulate) one or more aspects of the immune system.The immune system functions to protect the organism from infection andfrom foreign antigens by cellular and humoral mechanisms involvinglymphocytes, macrophages, and other antigen-presenting cells thatregulate each other by means of multiple cell-cell interactions and byelaborating soluble factors, including lymphokines and antibodies, thathave autocrine, paracrine, and endocrine effects on immune cells.

“Immune disorder” refers to abnormal functioning of the immune system.Immune disorders can be caused by deficient immune responses (e.g., HIV,AIDS) or overactive immune responses (e.g., allergy, auto-immunedisorders). Immune disorders can result in the uncontrolledproliferation of immune cells, uncontrolled response to foreign antigensor organisms leading to allergic or inflammatory diseases, aberrantimmune responses directed against host cells leading to auto-immuneorgan damage and dysfunction, or generalized suppression of the immuneresponse leading to severe and recurrent infections. Immune disorderrefers to disorders of the innate immune system (innate immunity) andthe adaptive immune system (adaptive immunity) Innate immunity refers toan early system of defense that depends on invariant receptorsrecognizing common features of pathogens. The innate immune systemprovides barriers and mechanisms to inhibit foreign substances, inparticular through the action of macrophages and neutrophils. Theinflammatory response is considered part of innate immunity. The innateimmune system is involved in initiating adaptive immune responses andremoving pathogens that have been targeted by an adaptive immuneresponse. However, innate immunity can be evaded or overcome by manypathogens, and does not lead to immunological memory. Adaptive immunityrefers to the ability to recognize pathogens specifically and to provideenhanced protection against reinfection due to immunological memorybased on clonal selection of lymphocytes bearing antigen-specificreceptors. A process of random recombination of variable receptor genesegments and the pairing of different variable chains generates apopulation of lymphocytes, each bearing a distinct receptor, forming arepertoire of receptors that can recognize virtually any antigen. If thereceptor on a lymphocyte is specific for a ubiquitous self antigen, thecell is normally eliminated by encountering the antigen early in itsdevelopment. Adaptive immunity is normally initiated when an innateimmune response fails to eliminate a new infection, and antigen andactivated antigen-presenting cells are delivered to draining lymphoidtissues. When a recirculating lymphocyte encounters its specific foreignantigen in peripheral lymphoid tissues, it is induced to proliferate andits progeny then differentiate into effector cells that can eliminatethe infectious agent. A subset of these proliferating lymphocytesdifferentiate into memory cells, capable of responding rapidly to thesame pathogen if it is encountered again.

Immune disorders caused by an impaired or immunocompromised immunesystem can produce a deficient immune response that leaves the bodyvulnerable to various viral, bacterial, or fungal opportunisticinfections. Causes of immune deficiency can include various illnessessuch as viruses, chronic illness, or immune system illnesses. Diseasescharacterized by an impaired immune system include, but are not limitedto, HIV/AIDS and severe combined immunodeficiency syndrome (SCIDS).

Immune disorders caused by an excessive response by the immune system.This excessive response can be an excessive response to one or moreantigens on a pathogen, or to an antigen that would normally be ignoredby the immune system. Diseases characterized by an overactive immunesystem include, but are not limited to, arthritis, allergy, asthma,pollinosis, atopy, and auto-immune diseases.

“Arthritis” refers to inflammation of the joints that can be caused,inter alia, by wear and tear on joints, or auto-immune attack onconnective tissues, or exposure to an allergen, e.g., as inadjuvant-induced arthritis. Arthritis is often associated with, orinitiated by, deposition of antibody-antigen complexes in jointmembranes and activation of an inflammatory response. Sometimes theimmune response is initiated by cells rather than antibodies, where thecells can produce a deposit in the joint membrane.

“Allergy” refers to an immune reaction to a normally innocuousenvironmental antigen (allergen), resulting from the interaction of theantigen with antibodies or primed T cells generated by prior exposure tothe same antigen. Allergy is characterized by immune and inflammatoryaspects, as the allergic reaction is triggered by binding of the antigento antigen-specific IgE antibodies bound to a high-affinity IgE receptoron mast cells, which leads to antigen-induced cross-linking of IgE onmast cell surfaces, causing the release of large amounts of inflammatorymediators such as histamine. Later events in the allergic responseinvolve leukotrienes, cytokines, and chemokines, which recruit andactivate eosinophils and basophils. The late phase of this response canevolve into chronic inflammation, characterized by the presence ofeffector T cells and eosinophils, which is most clearly seen in chronicallergic asthma.

“Asthma” refers to a chronic inflammatory disorder affecting thebronchial tubes, usually triggered or aggravated by allergens orcontaminants. Asthma is characterized by constriction of the bronchialtubes, producing symptoms including, but not limited to, cough,shortness of breath, wheezing, excess production of mucus, and chestconstriction

“Atopy” refers to the tendency to develop so-called “classic” allergicdiseases such as atopic dermatitis, allergic rhinitis (hay fever), andasthma, and is associated with a capacity to produce an immunoglobulin E(IgE) response to common allergens. Atopy is often characterized by skinallergies including but not limited to eczema, urticaria, and atopicdermatitis. Atopy can be caused or aggravated by inhaled allergens, foodallergens, and skin contact with allergens, but an atopic allergicreaction may occur in areas of the body other than where contact withthe allergan occurred. A strong genetic (inherited) component of atopyis suggested by the observation that the majority of atopic dermatitispatients have at least one relative who suffers from eczema, asthma, orhay fever.

“Pollinosis,” “hay fever,” or “allergic rhinitis,” are terms that referto an allergy characterized by sneezing, itchy and watery eyes, a runnynose and a burning sensation of the palate and throat. Often seasonal,pollinosis is usually caused by allergies to airborne substances such aspollen, and the disease can sometimes be aggravated in an individual byexposure to other allergens to which the individual is allergic.

“Auto-immune” refers to an adaptive immune response directed at selfantigens. “Auto-immune disease” refers to a condition wherein the immunesystem reacts to a “self” antigen that it would normally ignore, leadingto destruction of normal body tissues. Auto-immune disorders areconsidered to be caused, at least in part, by a hypersensitivityreaction similar to allergies, because in both cases the immune systemreacts to a substance that it normally would ignore. Auto-immunedisorders include, but are not limited to, Hashimoto's thyroiditis,pernicious anemia, Addison's disease, type I (insulin dependent)diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease,alopecia greata, anklosing spondylitis, antiphospholipid syndrome,auto-immune hemolytic anemia, auto-immune hepatitis, auto-immune innerear disease, auto-immune lymphoproliferative syndrome (ALPS),auto-immune thrombocytopenic purpura (ATP), Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome immune deficiency syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinindisease, CREST syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Guillain-Barre syndrome,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, juvenile arthritis, Meniere's disease, mixedconnective tissue disease, pemphigus vulgaris, polyarteritis nodosa,polychondritis, polyglancular syndromes, polymyalgia rheumatica,polymyositis, primary agammaglobulinemia, primary biliary cirrhosis,psoriasis, Raynaud's phenomenon, rheumatic fever, sarcoidosis,scleroderma, stiff-man syndrome, Takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis,vitiligo, and Wegener's granulomatosis.

“Collagen disease” or “connective tissue disease” refers to a chronicinflammatory auto-immune disorder in which autoantibodies attackcollagen found throughout the body. Connective tissues are composed oftwo major structural protein molecules, collagen and elastin; incollagen disease, autoantibodies directed against collagen will damageboth collagen and elastin due to the resulting inflammation. Collagendiseases include, but are not limited to, lupus erythematosus, Sjogren'ssyndrome, scleroderma, dermatomyositis, and polyarteritis nodosa.Rheumatoid-collagen disease refers to a disorder affecting theconnective tissue, with “rheumatic” symptoms including muscle stiffness,soreness and pain in the joints and associated structures.

“Inflammatory response” or “inflammation” is a general term for thelocal accumulation of fluid, plasma proteins, and white blood cellsinitiated by physical injury, infection, or a local immune response.Inflammation is an aspect of many diseases and disorders, including butnot limited to diseases related to immune disorders, viral infection,arthritis, auto-immune diseases, collagen diseases, allergy, asthma,pollinosis, and atopy. Inflammation is characterized by rubor (redness),dolor (pain), calor (heat) and tumor (swelling), reflecting changes inlocal blood vessels leading to increased local blood flow which causesheat and redness, migration of leukocytes into surrounding tissues(extravasation), and the exit of fluid and proteins from the blood andtheir local accumulation in the inflamed tissue, which results inswelling and pain, as well as the accumulation of plasma proteins thataid in host defense. These changes are initiated by cytokines producedby activated macrophages. Inflammation is often accompanied by loss offunction due to replacement of parenchymal tissue with damaged tissue(e.g., in damaged myocardium), reflexive disuse due to pain, andmechanical constraints on function, e.g., when a joint swells duringacute inflammation, or when scar tissue bridging an inflamed jointcontracts as it matures into a chronic inflammatory lesion.

“Anti-inflammatory” refers to the ability of a compound of the presentinvention to prevent or reduce the inflammatory response, or to sootheinflammation by reducing the symptoms of inflammation such as redness,pain, heat, or swelling.

Inflammatory responses can be triggered by injury, for example injury toskin, muscle, tendons, or nerves. Inflammatory responses can also betriggered as part of an immune response. Inflammatory responses can alsobe triggered by infection, where pathogen recognition and tissue damagecan initiate an inflammatory response at the site of infection.Generally, infectious agents induce inflammatory responses by activatinginnate immunity. Inflammation combats infection by delivering additionaleffector molecules and cells to augment the killing of invadingmicroorganisms by the front-line macrophages, by providing a physicalbarrier preventing the spread of infection, and by promoting repair ofinjured tissue. “Inflammatory disorder” is sometimes used to refer tochronic inflammation due to any cause.

Diseases characterized by inflammation of the skin, often characterizedby skin rashes, include but are not limited to dermatitis, atopicdermatitis (eczema, atopy), contact dermatitis, dermatitisherpetiformis, generalized exfoliative dermatitis, seborrheicdermatitis, drug rashes, erythema multiforme, erythema nodosum,granuloma annulare, poison ivy, poison oak, toxic epidermal necrolysisand roseacae.

Inflammation triggered by various kinds of injuries to muscles, tendonsor nerves caused by repetitive movement of a part of the body aregenerally referred to as repetitive strain injury (RSI). Diseasescharacterized by inflammation triggered by RSI include, but are notlimited to, bursitis, carpal tunnel syndrome, Dupuytren's contracture,epicondylitis (e.g. “tennis elbow”), “ganglion” (inflammation in a cystthat has formed in a tendon sheath, usually occurring on the wrist)rotator cuff syndrome, tendinitis (e.g., inflammation of the Achillestendon), tenosynovitis, and “trigger finger” (inflammation of the tendonsheaths of fingers or thumb accompanied by tendon swelling).

It is understood that the terms “immune disorder” and “inflammatoryresponse” are not exclusive. It is understood that many immune disordersinclude acute (short term) or chronic (long term) inflammation. It isalso understood that inflammation can have immune aspects and non-immuneaspects. The role(s) of immune and nonimmune cells in a particularinflammatory response may vary with the type of inflammatory response,and may vary during the course of an inflammatory response. Immuneaspects of inflammation and diseases related to inflammation can involveboth innate and adaptive immunity. Certain diseases related toinflammation represent an interplay of immune and nonimmune cellinteractions, for example intestinal inflammation (Fiocchi et al., 1997,Am J Physiol Gastrointest Liver Physiol 273: G769-G775), pneumonia (lunginflammation), or glomerulonephritis.

It is further understood that many diseases are characterized by both animmune disorder and an inflammatory response, such that the use ofdiscrete terms “immune disorder” or “inflammatory response” is notintended to limit the scope of use or activity of the compounds of thepresent invention with respect to treating a particular disease. Forexample, arthritis is considered an immune disorder characterized byinflammation of joints, but arthritis is likewise considered aninflammatory disorder characterized by immune attack on joint tissues.Thus, the observation that a compound of the invention reduces theinflammation seen in an animal model of arthritis, does not limit theobserved activity of the compound to anti-inflammatory activity. In adisease having both immune and inflammatory aspects, merely measuringthe effects of a compound of the present invention on inflammation doesnot exclude the possibility that the compound may also haveimmune-modulating activity in the same disease. Likewise, in a diseasehaving both immune and inflammatory aspects, merely measuring theeffects of a compound of the present invention on immune responses doesnot exclude the possibility that the compound may also haveanti-inflammatory activity in the same disease.

As used herein, the terms “peptide,” “polypeptide” and “protein” areused interchangeably and refer to two or more amino acids covalentlylinked by an amide bond or non-amide equivalent. The peptides of theinvention can be of any length. For example, the peptides can have fromabout two to about 100 or more residues, such as, 5 to 12, 12 to 15, 15to 18, 18 to 25, 25 to 50, 50 to 75, 75 to 100, or more in length.Preferably, peptides are from about 2 to about 18 residues. The peptidesof the invention include 1- and d-isomers, and combinations of l- andd-isomers. The peptides can include modifications typically associatedwith post-translational processing of proteins, for example, cyclization(e.g., disulfide or amide bond), phosphorylation, glycosylation,carboxylation, ubiquitination, myristylation, or lipidation.

Peptides disclosed herein further include compounds having amino acidstructural and functional analogues, for example, peptidomimetics havingsynthetic or non-natural amino acids or amino acid analogues, so long asthe mimetic has one or more functions or activities of compounds of theinvention. The compounds of the invention therefore include “mimetic”and “peptidomimetic” forms.

The terms “mimetic,” “peptide mimetic” and “peptidomimetic” are usedinterchangeably herein, and generally refer to a peptide, partialpeptide or non-peptide molecule that mimics the tertiary bindingstructure or activity of a selected native peptide or protein functionaldomain (e.g., binding motif or active site). These peptide mimeticsinclude recombinantly or chemically modified peptides, as well asnon-peptide agents such as small molecule drug mimetics, as furtherdescribed below.

In one embodiment, the PIF peptides of the invention are modified toproduce peptide mimetics by replacement of one or more naturallyoccurring side chains of the 20 genetically encoded amino acids (or Damino acids) with other side chains, for instance with groups such asalkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7 membered alkyl, amide, amidelower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy andthe lower ester derivatives thereof, and with 4-, 5-, 6-, to 7 memberedheterocyclics. For example, proline analogs can be made in which thering size of the proline residue is changed from 5 members to 4, 6, or 7members. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or nonaromatic. Heterocyclic groups cancontain one or more nitrogen, oxygen, and/or sulphur heteroatoms.Examples of such groups include the furazanyl, furyl, imidazolidinyl,imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g.1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl. Theseheterocyclic groups can be substituted or unsubstituted. Where a groupis substituted, the substituent can be alkyl, alkoxy, halogen, oxygen,or substituted or unsubstituted phenyl. Peptidomimetics may also haveamino acid residues that have been chemically modified byphosphorylation, sulfonation, biotinylation, or the addition or removalof other moieties.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24, 243-252, 1989). Certain peptidomimetic compounds are basedupon the amino acid sequence of the peptides of the invention. Often,peptidomimetic compounds are synthetic compounds having athree-dimensional structure (i.e. a “peptide motif”) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the peptidomimetic compound with the desired biologicalactivity, i.e., binding to PIF receptors, wherein the binding activityof the mimetic compound is not substantially reduced, and is often thesame as or greater than the activity of the native peptide on which themimetic is modeled. Peptidomimetic compounds can have additionalcharacteristics that enhance their therapeutic application, such asincreased cell permeability, greater affinity and/or avidity andprolonged biological half-life.

Peptidomimetic design strategies are readily available in the art (see,e.g., Ripka & Rich, Curr. Op. Chem. Biol. 2, 441-452, 1998; Hruby etal., Curr. Op. Chem. Biol. 1, 114-119, 1997; Hruby & Balse, Curr. Med.Chem. 9, 945-970, 2000). One class of peptidomimetics a backbone that ispartially or completely non-peptide, but mimics the peptide backboneatom-for atom and comprises side groups that likewise mimic thefunctionality of the side groups of the native amino acid residues.Several types of chemical bonds, e.g., ester, thioester, thioamide,retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, areknown in the art to be generally useful substitutes for peptide bonds inthe construction of protease-resistant peptidomimetics. Another class ofpeptidomimetics comprises a small non-peptide molecule that binds toanother peptide or protein, but which is not necessarily a structuralmimetic of the native peptide. Yet another class of peptidomimetics hasarisen from combinatorial chemistry and the generation of massivechemical libraries. These generally comprise novel templates which,though structurally unrelated to the native peptide, possess necessaryfunctional groups positioned on a nonpeptide scaffold to serve as“topographical” mimetics of the original peptide (Ripka & Rich, 1998,supra).

The PIF assay, as disclosed in U.S. Pat. No. 5,646,003 to Barnea et al.,entitled “Preimplantation Factor” issued Jul. 9, 1997, and in U.S. Pat.No. 5,981,198 to Barnea et al., entitled “Preimplantation Factor”granted Nov. 9, 1999, the disclosures of which are incorporated hereinby reference in their entirety, may be used to measure the response ofthe immune system to pregnancy specific preimplantation factors. Studiesemploying the PIF assay for culture media of human or mouse embryos thatwere grown, show that PIFs were able to increase the in-vitro formationof rosettes between donor lymphocytes and platelets in the presence ofmonoclonal anti-CD2 (type T11-1). Lymphocyte-platelet rosettes resultfrom the interaction of the T cell surface protein CD2 with its ligandCD58 expressed on the platelet membrane. Anti-CD2, by binding to the CD2antigen on the T cells, inhibits their interaction with platelets.However, the embryo-derived factor(s), PIFs, present in the culturemedium or pregnant peripheral sera appears to counteract thisinhibition. The PIF activity was already apparent in the viable two-cellstage embryo. Thus both of those compounds properties are very likely tobe similar. This observation strongly suggests that there are severalputative compounds that may be very potent, and create an environmentthat is favorable for pregnancy.

Using this assay, it has been determined that the presence of PIFactivity in maternal serum within four days after embryo transferindicates a >70% chance of successful pregnancy outcome. In contrast,absence of PIF activity indicated that pregnancy would not develop in97% of cases. PIF is detectable 5-6 days after intrauterine inseminationand is absent in non-pregnant serum and in culture media of non-viableembryos, present in the sera of various mammals including horse, cow,pig and humans. Without wishing to be bound by theory, the PIF assayresults indicate that if the embryo is able to secrete theseimmunomodulatory PIF compounds, it is capable of implanting andachieving a good pregnancy outcome. The importance of PIF as a marker ofa good quality pregnancy is further illustrated by the fact that if apregnancy ends in miscarriage, the PIF activity progressively declinesuntil it reaches non-detectable levels. In contrast, in the case of apoor quality pregnancy, Human Chronic Gonadotropin (hCG) levels do notchange significantly for the next 3 weeks until the miscarriage isclinically evident.

PIF activity is found in several mammalian species, including humans,horse, cow, pig, and mouse and sheep. Human immune cells used for thePIF assay (homologous lymphocytes and platelets) interacted well withthe human sera, as well as with sera from different species and embryoculture media. This cross-species interaction indicates that similarcompounds are involved in the different species. PIF activity is due tothe presence of similar low molecular weight peptides, both in mouseembryo culture media and in pregnant porcine serum. A PIF assay was usedas a test to identify and characterize the PIF related compounds withina conditioned mouse embryo culture media. Using a multi-stepchromatographic technique, coupled with the PIF bioassay, a group of aputative PIF embryo derived peptides with 9-18 amino acids in lengthwere identified and sequenced. These sequences are disclosed inPCT/US02/20599 to Barnea et al., entitled “New Assays forPreimplantation Factor and Preimplantation Peptides,” filed Jun. 28,2002, the contents of which are incorporated herein by reference intheir entirety. Based on the sequences derived, synthetic peptides weregenerated.

The first natural PIF compound identified, termed nPIF-1₍₁₅₎ (SEQ IDNO:1), is a 15 amino acid peptide. A synthetic version of this peptide,BPIF-1₍₁₅₎ (SEQ ID NO:13), showed activity that was similar to thenative peptide, nPIF-1₍₁₅₎ (SEQ ID NO:1). This peptide is homologous toa small region of the Circumsporozoite protein, a malaria parasite. Thesecond PIF peptide, nPIF-2₍₁₃₎ (SEQ ID NO:7), includes 13 amino acidsand shares homology with a short portion of a large protein namedthyroid and retinoic acid transcription co-repressor, which isidentified as a receptor-interacting factor, (SMRT); the syntheticversion is sPIF-2 (SEQ ID NO:14). The third distinct peptide, nPIF-3₍₁₈₎(SEQ ID NO:10), consists of 18 amino acids and matches a small portionof reverse transcriptase; the synthetic version of this peptideBPIF-3₍₁₈₎ is (SEQ ID NO:15). nPIF-4₍₉₎ (SEQ ID NO:12) shares homologywith a small portion of reverse transcriptase.

A list of PIF peptides, both natural and synthetic, are provided belowin Table 1. Antibodies to various PIF peptides and scrambled PIFpeptides are also provided.

TABLE 1  PIF Peptides (SEQ ID NO) Peptide Amino Acid SequenceSEQ ID NO: 1 nPIF-1₁₅ MVRIKPGSANKPSDD isolated native, matches region ofCircumsporozoite protein (Malaria) SEQ ID NO: 2 nPIF-1_((15-alter))MVRIKYGSYNNKPSD isolated native, matches region ofCircumsporozoite protein (Malaria) SEQ ID NO: 3 nPIF-1₍₁₃₎ MVRIKPGSANKPSisolated native, matches region of Circumsporozoite protein (Malaria)SEQ ID NO: 4 nPIF-1₍₉₎ MVRIKPGSA isolated native, matches region ofCircumsporozoite protein (Malaria) SEQ ID NO: 5 scrPIF-1₁₅GRVDPSNKSMPKDIA synthetic, scrambled amino acid sequencefrom region of Circumsporozoite protein Malaria SEQ ID NO: 6 nPIF-2₍₁₀₎SQAVQEHAST isolated native, matches region of humanretinoid and thyroid hormone receptor-SMRT SEQ ID NO: 7 nPIF-2₍₁₃₎SQAVQEHASTNMG isolated native, matches region of humanretinoid and thyroid hormone receptor (SMRT) SEQ ID NO: 8 scrPIF-2₍₁₃₎EVAQHSQASTMNG synthetic, scrambled amino acid sequencefrom region of human retinoid and thyroid hormone receptor SMRTSEQ ID NO: 9 scrPIF-2₍₁₄₎ GQASSAQMNSTGVH SEQ ID NO: 10 nPIF-3₍₁₈₎SGIVIYQYMDDRYVGSDL isolated native, matches region of Rev TransSEQ ID NO: 11 Neg control GMRELQRSANKsynthetic, scrambled amino acid sequence forfrom region of Circumsporozoite protein negPIF-1₍₁₅₎ MalariaSEQ ID NO: 12 nPIF-4₍₉₎ VIIIAQYMDisolated native, matches region of Rev Transantibody of native isolated nPIF-1₁₅ AbPIF-1₍₁₅₎ (SEQ ID NO: 13)sPIF-1₍₁₅₎ MVRIKPGSANKPSDD synthetic, amino acid sequence from regionof Circumsporozoite protein Malaria (SEQ ID NO: 14) sPIF-2₍₁₃₎SQAVQEHASTNMG synthetic, amino acid sequence from of humanretinoid and thyroid hormone receptor SMRT (SEQ ID NO: 15) sPIF-3₍₁₈₎SGIVIYQYMDDRYVGSDL synthetic, amino acid sequence from regionof Circumsporozoite protein Malaria (SEQ ID NO: 16) sPIF-1₍₉₎ MVRIKPGSAsynthetic, amino acid sequence from regionof Circumsporozoite protein Malariaantibody of native isolated nPIF-2₍₁₃₎ AbPIF-2₍₁₃₎antibody of native isolated nPIF-3₍₁₈₎ AbPIF-3₍₁₈₎ (SEQ ID NO: 17)sPIF-4₍₉₎ VIIIAQYMD synthetic SEQ ID NO: 18 sPIF-1₍₅₎ MVRIK syntheticSEQ ID NO: 19 sPIF-1₍₄₎ PGSA synthetic n = native, s = synthetic, scr =scrambled, same AA, ( ) = number of AA, Ab = antibody

In another embodiment, a method of detecting a PIF petptide is provided.The PIF peptide may include, for example, SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17. In a further embodiment, a method of detecting a PIF peptide whichincludes a fragment of nPIF-1₁₅, nPIF-2₁₃, nPIF-3₁₈ or nPIF-4₉.

PIF peptides and peptidemimetics of the present invention may be coupledto produce labeled peptides, for example but not limited to FITC,biotin, rhodamine, radioactive labels, fluorescent nanocrystals, andother labels known to those skilled in the art, that may be used toidentify PIF receptor sites present on immune cells, endometrium, on theembryo itself, as well as elsewhere within the body where PIF peptidesspecifically bind.

Embodiments of the present invention may be used to identify and clonethe genes that are responsible for PIF peptides expression. cDNA libraryis prepared from human placenta (Invitrogen) that have libraries of1-2.5 kb size inserts which represent even the rarest sequences.Oligonucleotides are generated based on the peptides sequences and areprobed against the cDNA library using plate screening procedures. ThePIF peptide presence in the placenta was adding previously documentedusing immunohistochemical techniques by labeled PIF-1 antibody. Thespecies of PIF peptides present in the placenta are confirmed withaffinity purified and labeled PIF-1, PIF-2, and PIF-3 antibodies using aWestern blot. The present invention may be used to generate specificantibodies polyclonal and monoclonal for assay development to measurePIF levels and activity in biologic fluids and tissues such as but notlimited to serum, blood, urine, milk, and saliva as well as embryoculture media, gestational tissue, and fetal tissue.

Another embodiment of the present invention provides for makingpolyclonal or monoclonal antibodies that were raised against PIF. In onenon-limiting embodiment, polyclonal or monoclonal antibodies may beraised against PIF in mice and rabbits. In another embodiment,antibodies to PIF may be created by providing a hybridoma cell thatproduces a monoclonal antibody specific for a PIF peptide and culturingthe cell.

Such antibodies provide a method for determining the presence of PIFlevels in samples by using but not limited to ELISA, EIA, lateral flowassay, microfluidics or mass spectometry. Such a method and antibodiesmay allow precise measurements of PIF levels in fluids such as but notlimited to maternal blood, urine, saliva, milk, and embryo culture mediaand gestational tissues. The method is applicable for all PIF peptidesand may be used to provide an early diagnostic method that reflectspregnancy and its viability in various patients starting at thepre-implantation period. The patients may include women, to monitorresults of infertility therapy and pregnancy well being, as well asother mammals, including farm and non-farm animals, and non-mammals. Inthe embryo culture media the ELISA assay using such antibodies providesa method for assessing the presence of PIF peptides to assess embryoviability before transfer. Various aspects of the present invention willbe illustrated with reference to the following non-limiting examples.

In one embodiment of the present invention, a PIF peptide is provided.Such PIF peptides may be useful for treating or amelioratingimmune-mediated disorders, such as autoimmune diseases.

In another embodiment, a pharmaceutical composition comprising a PIFpeptide is provided. In preferred embodiments, the pharmaceuticalcomposition comprises an effective amount of a PIF peptide.

In another embodiment, a method of treating or preventingimmune-mediated disorders is provided. In a preferred embodiment, themethod comprises administering an effective amount of a PIF peptide to asubject in need thereof. The methods are particularly useful in treatingor preventing immune-mediated disorders, including, but not limited to,graft-versus-host disease, type 1 diabetes, multiple sclerosis,ulcerative colitis, Crohn's disease, rheumatoid arthritis and the like.

In a further embodiment, a method for treating or preventingimmune-mediated disorders comprising administering an effective amountof a PIF peptide in combination with one or more immunotherapeutic drugsto a subject in need thereof is provided. Such a combination may enhancethe effectiveness of the treatment of either component alone, or mayprovide less side effects and/or enable a lower dose of eithercomponent.

The present data demonstrate that short-term exposure to PIF-1 at lowdoses is associated with a long-term protection against development ofautoimmune disorders of disparate origin. While not wishing to be boundby theory, based on the currently understood aspects of PIF-1'smechanism of action, the peptide appears to act independently of thetype of pathophysiological features of the autoimmune disease examinedaddressing them in an etiology-independent manner. This agrees with theproperties of PIF-1 following examination of its effects on PBMC. PIF-1was found to have widespread modulatory effects on cellular immunology,as well as on cytokine production and secretion acting through specificinducible receptors present on subtypes of PBMC. PIF-1 appears to affectdisparate aspects of immunity since it responds to various mitogenchallenge, PHA, CD3MAb, CD3MAb/CD28MAb, and MLR. PIF-1 exposure blocksactivated, but not basal, PBMC proliferation. In addition while therewas some bias towards T_(H)2, PIF-1 caused an increase in both T_(H)1and T_(H)2 cytokines following mitogen exposure. This may indicate thatPIF-1 helps to maintain the balance between the two immune modalities,not allowing either extreme inflammation or immune suppression. Byblocking activated, but not basal, immunity the ability to respond to animmunogenic challenge such as pathogen exposure, and/or maternalrejection is well maintained. This may explain the significant efficacyobserved in the current mouse studies. Overall, PIF-1's mechanism ofaction is distinct from other currently used immune suppressive agents.

The three autoimmune models studied are quite distinct: BMT replicatesexposure of the model organism to foreign immune cells as would be thecase in GVHD; NOD replicates Type 1 diabetes induced by a specificattack on the pancreas by transplanted foreign T cells; and EAE modelsMS by stimulating a bacterial toxin and protein immunogen attack on thenervous tissue of the brain. However, all of the models arecharacterized by an induced immune response against the host organismand, consequently, to the autoimmune-induced destruction of vital organsand, ultimately, death. PIF-1 appears to successfully neutralize theinitiation of this cascade irrespective of the initiating insult. Thiscan be analogized to pregnancy, in which the embryo is tolerated by themother, but the mother remains able to respond adequately to pathogens.Pregnancy may also leave the mother less susceptible to autoimmunity andmalignancy, to some degree. Therefore, recreating an environment whereselect cells are tolerated while pathogens are attacked in anon-pregnancy setting may be the central mechanism of PIF-1's action inautoimmune model systems.

In all three models, efficacy was obtained in low doses, 0.1-1mg/kg/day. By contrast, somewhat lower efficacy is observed at higherdosing in two models (NOD 2.73 mg/kg/day and BMT 5 mg/kg/day). Thisagrees with in vitro data where maximal PIF-1 efficacy was found at 1-50nM concentrations, while higher doses were either less effective or noteffective at all. This further suggests a receptor-dependent mechanismof action that is mostly responsive at a narrow range of concentrationsand otherwise may be down-regulated when concentrations are raisedbeyond optimal levels. These observations strongly indicate that PIFexerts this biphasic effect through physiological and notpharmacological mechanisms.

While the mechanism for the long-term protective effect of PIF-1 as seenin these autoimmune models is not clear, and without wishing to be boundby theory, it appears that PIF-1 initiates, following mitogen exposure,a time-dependent block of proliferation and leads to a cascade of T_(H)1and T_(H)2 cytokine secretion, some being secreted earlier while others,later. Such sequential effects may lead to a long-term modification ofthe immune environment.

PIF-1 appears to act through putatively novel receptors that arepredominantly expressed on monocytes and macrophages. However, whenstimulated by mitogens, the expression of these receptors becomessignificant on T and B Cells but not NK cells. Differences in theexpression pattern of PIF-1 receptors may explain the differences in theresponse induced by PIF-1 seen in un-stimulated and stimulatedenvironments. In an un-stimulated environment, PIF-1 may only have a lowlevel of activity on T and B Cells. However, activation of the immunesystem in response to an immune system challenge may lead to theexpression of the PIF-1 receptors on T and B Cells initiating long termtolerance.

PIF-1's action appears to be independent of TCR, calcium-channels or PKCpathways, mechanisms through which most immune-suppressive agents act,and CD4+/CD25+ cells (T reg) cells that are of relevance in variousautoimmune diseases. On the other hand, PIF-1's action may involveNFAT-1 suppression.

In pregnancy, embryo viability is dependent on maternal tolerance of theembryo, but there is a clear time lag between embryo expulsion bymiscarriage and reduction of PIF levels in maternal circulation. Infact, PIF disappears from maternal circulation up to three weeks beforematernal human chorionic gonadotropin (hCG) levels dropped andmiscarriage ensues. Perhaps, a similar mechanism is involved inmaintaining tolerance, or protective effects against autoimmunity longterm, as is documented in the three model systems tested followingcessation of therapy.

In a preliminary study, the effect of PIF-1 administration using anAlzet® pump for 7 days after mating on implantation rates in mice wasexamined. As expected, PIF-1 did not exert any adverse effects.Moreover, PIF-1 may have actually increased the rates of fetal survivalvs. control by day 13 of pregnancy, as documented at the time ofCeasarean section. This data combined with the additional six animaltrials provides a strong support for the lack of PIF-1 toxicity.

We also found that FITC-PIF-1 injected IV in mice accumulated in thespleen and was cleared from circulation into the kidney within minutes.This shows that PIF-1 specifically targets immune cells of the spleen invivo, and has a short half-life in circulation. The long term effect ofPIF administration may reflect a pharmacodynamic type of mechanism sincethe peptide has a short half-life, rather than a pharmacologic effectthat is produced while the drug is given and is dependent on the levelsof the drug in the circulation.

The observations that PIF-1 exerts long-term protection after short-termexposure in all three models tested raises the possibility that PIF-1therapy could be used for long-term management of patients withautoimmune diseases, perhaps initially using an insulin pump that couldreplicate the function of an Alzet® pump followed by periodic PIF-1administration, over a long term. Other devices capable of continuousand/or long term administration may also be useful. In addition, it maybe possible to develop an increased half-life, modified peptide (such asby PEGylation) and/or to use transdermal delivery system for long term,but minimally invasive use. Finally, due to PIF-1's simple structure andsmall size, in which shorter versions of the peptide are similarlyeffective (at least in vitro), oral delivery may become possible, whichcould transform PIF-1 into a convenient chronic therapy.

Ultimately, a novel embryo-derived peptide, PIF, creates a tolerogenicstate at low doses following short-term treatment leading to long-termprotection in several distinct severe autoimmune models. This effect isexerted without apparent toxicity.

For therapeutic treatment of the specified indications, a PIF peptidemay be administered as such, or can be compounded and formulated intopharmaceutical compositions in unit dosage form for parenteral,transdermal, rectal, nasal, local intravenous administration, or,preferably, oral administration. Such pharmaceutical compositions areprepared in a manner well known in the art and comprise at least oneactive PIF peptide associated with a pharmaceutically carrier. The term“active compound”, as used throughout this specification, refers to atleast one compound selected from compounds of the formulas orpharmaceutically acceptable salts thereof.

In such a composition, the active compound is known as “activeingredient.” In making the compositions, the active ingredient willusually be mixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier that may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semisolid, or liquid material that acts as a vehicle, excipientof medium for the active ingredient. Thus, the composition can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,emulsions, solutions, syrups, suspensions, soft and hard gelatincapsules, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate alginates, calcium salicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup,methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesiumstearate, water, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Thecompositions may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art.

For oral administration, a compound can be admixed with carriers anddiluents, molded into tablets, or enclosed in gelatin capsules. Themixtures can alternatively be dissolved in liquids such as 10% aqueousglucose solution, isotonic saline, sterile water, or the like, andadministered intravenously or by injection.

The local delivery of inhibitory amounts of active compound for thetreatment of immune disorders can be by a variety of techniques thatadminister the compound at or near the targeted site. Examples of localdelivery techniques are not intended to be limiting but to beillustrative of the techniques available. Examples include localdelivery catheters, site specific carriers, implants, direct injection,or direct applications, such as topical applicaiton.

Local delivery by an implant describes the surgical placement of amatrix that contains the pharmaceutical agent into the affected site.The implanted matrix releases the pharmaceutical agent by diffusion,chemical reaction, or solvent activators.

For example, in some aspects, the invention is directed to apharmaceutical composition comprising a PIF peptide, and apharmaceutically acceptable carrier or diluent, or an effective amountof a pharmaceutical composition comprising a PIF peptide.

The compounds of the present invention can be administered in theconventional manner by any route where they are active. Administrationcan be systemic, topical, or oral. For example, administration can be,but is not limited to, parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, oral, buccal, ocularroutes, intravaginally, by inhalation, by depot injections, or byimplants. Thus, modes of administration for the compounds of the presentinvention (either alone or in combination with other pharmaceuticals)can be, but are not limited to, sublingual, injectable (includingshort-acting, depot, implant and pellet forms injected subcutaneously orintramuscularly), or by use of vaginal creams, suppositories, pessaries,vaginal rings, rectal suppositories, intrauterine devices, andtransdermal forms such as patches and creams.

Specific modes of administration will depend on the indication. Theselection of the specific route of administration and the dose regimenis to be adjusted or titrated by the clinician according to methodsknown to the clinician in order to obtain the optimal clinical response.The amount of compound to be administered is that amount which istherapeutically effective. The dosage to be administered will depend onthe characteristics of the subject being treated, e.g., the particularmammal or human treated, age, weight, health, types of concurrenttreatment, if any, and frequency of treatments, and can be easilydetermined by one of skill in the art (e.g., by the clinician).

Pharmaceutical formulations containing the compounds of the presentinvention and a suitable carrier can be solid dosage forms whichinclude, but are not limited to, tablets, capsules, cachets, pellets,pills, powders and granules; topical dosage forms which include, but arenot limited to, solutions, powders, fluid emulsions, fluid suspensions,semi-solids, ointments, pastes, creams, gels and jellies, and foams; andparenteral dosage forms which include, but are not limited to,solutions, suspensions, emulsions, and dry powder; comprising aneffective amount of a polymer or copolymer of the present invention. Itis also known in the art that the active ingredients can be contained insuch formulations with pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman& Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) can be consulted.

The compounds of the present invention can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. The compounds can be administered by continuous infusionsubcutaneously over a predetermined period of time. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

For oral administration, the compounds can be formulated readily bycombining these compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by adding a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, fillers such as sugars,including, but not limited to, lactose, sucrose, mannitol, and sorbitol;cellulose preparations such as, but not limited to, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with filler such as, e.g., lactose, binders such as, e.g.,starches, and/or lubricants such as, e.g., talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers can be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal administration, the compositions can take the form of, e.g.,tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds of the present invention can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds ofthe present invention can also be formulated as a depot preparation.Such long acting formulations can be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection.

Depot injections can be administered at about 1 to about 6 months orlonger intervals. Thus, for example, the compounds can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

In transdermal administration, the compounds of the present invention,for example, can be applied to a plaster, or can be applied bytransdermal, therapeutic systems that are consequently supplied to theorganism.

Pharmaceutical compositions of the compounds also can comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as, e.g., polyethylene glycols.

The compounds of the present invention can also be administered incombination with other active ingredients, such as, for example,adjuvants, or other compatible drugs or compounds where such combinationis seen to be desirable or advantageous in achieving the desired effectsof the methods described herein.

This invention and embodiments illustrating the method and materialsused may be further understood by reference to the followingnon-limiting examples.

Example 1

Peptide synthesis: Synthetic PIF-1₁₅ (MVRIKPGSANKPSDD) was obtained bysolid-phase peptide synthesis (Peptide Synthesizer, Applied Biosystems)employing Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Finalpurification is carried out by reverse-phase HPLC and identity isverified by MALDI-TOF mass spectrometry and amino acid analysis andpurified to >95%, by HPLC, and documented by mass spectrometry(Biosynthesis, Texas).

Mice: C57BL/6(H-2b) male and female and (C57BL/6xBALB/c) F1 (H-2d/b)male, five- to six-week-old mice (GVHD studies) and seven- to eight-weekold SJL mice (MS studies) were purchased from Harlan (Israel), and maleand female seven- to eight-week-old NOD mice were obtained from JacksonLaboratories (Maine). All mice were maintained under conditions approvedby the Institutional Animal Care and Use Committee of the HebrewUniversity in Jerusalem in accordance with the national laws andregulations for protection of animals.

GvHD model:Recipients (C57BL/6xBALB/c) F1 mice received lethalwhole-body irradiation by a single dose of 1000rad/dose and werereconstituted with 5−8×10⁶ C57BL/6 bone marrow (BM) cells and 10−20×10⁶spleen cells. BM from C57BL/6 donor mice was collected by flushing offemur, humerus and tibia into 10% FCS/PBS. BM mononuclear cells wereisolated from the interface after centrifugation on a Ficoll-Hipaquegradient. Spleens were crushed through 70 μm screens into 10% FCS/PBS.BM cells plus spleen cells were inoculated intravenously into whole-bodyirradiated mice one-day post radiation. PIF-1 therapy (0.1-1 mg/kg/day)was administered in three separate animal trials 5-10/group vs. controlby implanting under anesthesia an Alzet® pump in the dorsal subcutaneousregion at the day of transplant for one to two weeks providingcontinuous release of PIF-1. Evaluation of GvHD model animals wascarried out by examining body weight, skin lesions, animal survival, andhistological examination. Animal weight was examined every three daysfollowing BM transplantation, scoring for skin manifestations of GVHDwas carried out from day 12 post BMT up to four months. Skin and liversamples were fixed in 10% formalin embedded in paraffin and stained withhematoxylin and eosin and evaluated for ulcers, in the former andlymphocyte infiltration in the latter. Results were evaluated by χ² andANOVA.

Assay for chimerism: Mice were anesthetized and blood taken from theretro-orbital sinus of the eye. WBC (2-8×10⁵/sample) were separated,directly stained with anti-H-2K^(b)-FITC (IgG_(2a)) oranti-H-2K^(d)-FITC (IgG_(2a)) monoclonal antibodies (mAb) (Serotec,USA), and analyzed by FACS analysis (FACStar plus, Becton Dickinson, SanJose, Calif., USA). Background binding of each H-2K-specific mAb wasdetermined by staining with it the cells of non-relevant haplotype.

GVHD Model experiment I. Following low-burden BMT, GVHD development wasexamined following PIF-1 therapy (0.1-1 mg/kg/day) given for one weekusing an implanted Alzet® pump followed by one month observation. Of thePIF-1-treated mice, 0.1 or 1 mg/kg/day for one week, all eight mice didnot develop GVHD. Four out of five controls developed GVHD grade II-III(P<0.01). Mice following BMT and short term PIF-1 treatment remainedcompletely protected against the development of GVHD at one month aftercessation of therapy. In contrast, in control mice, severe skinulcerations and weight loss developed (mean mouse weight 21.9 g, highdose PIF-1 (N=3), 20.2 g low dose PIF-1 (N=5), and 19.5 g in controls).

GVHD Model experiment II (FIG. 1). We examined whether PIF-1 couldprevent GVHD development in a higher-burden BMT (double number of spleencells transplanted than the low-burden BMT). Following exposure to PIF-1(0.1-1 mg/kg/day) for two weeks, total protection against GVHD wasobtained within three weeks with the high dose therapy 7/7 vs. 9/9 incontrol with GVHD. This protection remained also significant (P<0.04) atthirty days post-BMT in both treatment groups, as evaluated by the GVHDscore in those which developed the disease (FIG. 2).

GVHD Model experiment III (FIG. 3). We examined whether short-termtreatment can lead to long term survival after cessation of therapy.Following high-burden BMT, PIF-1 1-5 mg/kg/day for two weeks wasadministered and mice were followed for an additional three and one-halfmonths without therapy. PIF-1 conferred a significant protection asdetermined by mouse survival at the end of the observation period. Sevenof nine of the PIF-1 treated mice survived compared to only two out often in controls (P<0.02). Higher-dose therapy was less effective,although it was still associated with a higher survival rate thancontrols (5/9 survived). Significant protection from weight loss wasalso achieved following PIF-1 1/mg/kg/day exposure vs. controls. Thiseffect became significant after day 33 from BMT. In control mice,following BMT, GVHD-induced skin ulcerations were observed. Short-termPIF-1 therapy prevented the development of such lesions in the longterm. Liver histology also documented that lymphocytic infiltrates,indicating autoimmune response, were noted in control, but not inPIF-1-treated mice one month after cessation of therapy. The degree ofBMT incorporation into the recipient mice was determined. Results showthat there was a quasy total incorporation of grafted bone marrow afterfive weeks after BMT (87.5±2.4), reflected a very high degree ofchimerism.

Allo-BMT followed destruction of the host's immune system by total bodyradiation. Thereby the BMT recipient is highly vulnerable to immuneattack by the transplanted foreign immune cells. Low dose (micromolar)PIF-1 administration totally prevented GVHD while therapy wasadministered. More remarkably, long-term protection after cessation oftherapy was obtained, as reflected by the significant prevention of GVHDdevelopment and long-term survival for several months vs. control mice.This effect was not associated with any toxicity, as documented by mouseweight, skin appearance, and skin and liver histology. This was alsodocumented by the significant degree of chimerism (about 90%) thatdeveloped in the peripheral PBMC within five weeks following BMT,indicating that at that time the great majority of the mice immunesystem was constituted of the transplanted BM.

PIF-1's long-term protective effect after cessation of therapy isparticularly significant, as other BMT therapies are effective onlyduring active administration. Furthermore, the current BMT modelinvolved a clear mismatch between the recipient and the donor, and largequantities of cells were transplanted, while clinical settings useclosely matched BM donors, which nevertheless often, up to 70% resultsin various degrees of GVHD.

Example 2

Materials and methods are the same as Example 1.

DM (adoptive transfer NOD) model. Male NOD mice were irradiated (650rad), and injected IV next day with 250 Mil spleen cells collected fromfemale NOD diabetic mice. PIF-1 was injected in two doses 0.83 mg/kg/day(N=5) and 2.73 mg/kg/day (N=7) for 28 days using an Alzet® pump,implanted subcutaneously providing continuous release of the peptide,followed by a 40-day observation period. Animals were monitored for DMdevelopment by determining fasting glucose levels in both blood andurine. Results were evaluated using ANOVA.

NOD diabetes model. We examined the effect of PIF-1 in a differentautoimmune model, NOD adoptive transfer. In this model, transfer ofdiabetic splenocytes from female to male mice leads progressively to thedevelopment of diabetes mellitus. Exposure to PIF-1 0.83-2.73 mg/kg/dayfor the first 28 days had a long-term protective effect against thedestruction of pancreatic cells and the consequent high serum glucoselevels. FIG. 4 shows a life table analysis of NOD mice followingadoptive transfer of splenocytes from a diabetic female mouse. By 70days, at the conclusion of the experiment, PIF-1 was totally protectivein 11/12 of mice treated with PIF-1 while 6/7 in the control group hasalready developed diabetes. Interestingly, the only PIF-1 treated mousethat developed DM received a higher treatment dose. The development ofdiabetes was documented by increased serum glucose levels; in certaincontrol animals it reached >600 mg/dl. Table 2, below, shows individualmice glucose levels after cessation of therapy.

TABLE 2 Non fasting blood Fasting blood NOD male glucose mg/dL (dayglucose mg/dL (day mice No. 40) 69) Control 1 >600 355 2 >600 377 3 202232 4 193 5 >600 6 298 207 7 135 123 PIF 0.83 mg/kg/ 1 131 97 day 2 112106 3 137 119 4 132 109 5 130 149 PIF 2.73 mg/kg/ 1 118 87 day 2 126 1083 125 111 4 113 144 5 158 514 6 123 103 7 115 109

In the control group, most mice developed diabetes by 40 days.Additionally, histological examination demonstrated that PIF treatedmice were protected against inflammation of the pancreas v. control(data not shown).

To further document PIF-1's immune-modulatory effects, we used the NODmouse adoptive transfer model, which results in the development ofdiabetes (reflected by high glucose levels) due to the destruction ofthe recipient's pancreas by transfer of autoreactive splenocytes from adiabetic mouse that targets specifically that organ. Using thisaggressive model, we documented a long-term protection against DMdevelopment using PIF-1 therapy. These results open the possibility ofexamining young adults that have recently developed DM in whom there hasnot been a total destruction of insulin-producing pancreas cells. Suchan early intervention could lead to a decreased need for insulinadministration, or even allow long-term oral anti-diabetic therapy.Since we found that PIF-1 targets isolated splenocytes that provides arationale for the protective effects that were observed. In TIDM primedT cells and macrophages directly attack the pancreas which is followedby local increase in T_(H)1 cytokines (i.e., TNF, interferon-) thatfurther amplify the auto-destructive process. PIF-1 may act on both ofthese aspects of autoimmunity by blocking activated immune cellsproliferation activation and modulating cytokines secretion, towards aT_(H)2 pattern (i.e., major increase in IL10).

Example 3

Materials and methods are the same as Example 1.

MS EAE model: experimental autoimmune encephalomyelitis, SJL mice 7-8weeks old were injected in the tail base with 1:1 of 200 μg proteolyticprotein peptide (PLP) together with 200 μg CFA and IFA (containingmycobacterium tuberculosis). On the same day and two days later, micewere injected IP with 250 ng pertussis toxin. Within nine days, animalsstarted developing paralysis. PIF-1 was administered using asubcutaneously implanted Alzet® pump at 0.75 mg/kg/day for 28 days andits effect was compared to a control group. Daily monitoring of thedegree of paralysis (grade 0/no disease-5/dead animal) occurred up to 40days. PIF-1's protective effects were calculated using the Mann-Whitneynon parametric test.

MS model. We further examined whether PIF-1 therapy could be effectivein an additional autoimmune model, experimental autoimmuneencephalomyelitis (EAE) in which the majority of the damage occurs inpoorly accessible region of the body, the central nervous system. Byexposing mice to a combination of a toxic agent (PLP) for the nervoussystem together with boosting further the inflammatory response with twoadditional types of bacterial-toxins led to rapid paralysis <10 days.The exposure to PIF-1 at 0.75 mg/kg/day for 28 days led to a continuousprotection by significantly reducing the paralysis score, as determinedby daily observations using a clinical score. The protective effect alsolasted for at least two weeks after stopping therapy (P<0.002).

The experimental myeloencephalitis, EAE, is recognized as a highlyrelevant and acute model for MS. The exposure to auto-antigens coupledby induction with two bacterial immunogens leads to progressiveparalysis within short term. We found that PIF-1 led to a significantreduction in the paralysis score across the observation period whichpersisted even two weeks after cessation of therapy. This is anindication that autoimmune neurological disorders may be alleviated byPIF-1. Additionally, histological examination demonstrated that PIFtreated mice were protected against inflammation of the spinal cord v.control (data not shown).

MS is believed to be the result of a genetic predisposition followed bya viral insult that leads to CD4+ autoreactive cells followed bydifferentiation to the T_(H)1 phenotype. On the other hand, local damageto central nervous system may occur by CD8+ T cells, and other elementsthat are involved in the innate immune system. This leads to alteredT_(H)2 cytokines, regulatory T, and NK cells and IFN secretion. We havepreviously shown that several elements of this immune cascade aremodulated by PIF-1, consequently, the tolerogenic peptide may beinvolved in one or more aspects of this immune disorder.

Example 4

To determine maximally tolerated dose of PIF-1 in patients who developGVHD after matched BMT, using an insulin pump. Recipients with grade IIGVHD will be randomized into three groups: (1) continue conventionaltherapy (i.e., steroids and cyclosporin A), as control; (2) add PIF-1therapy while continuing conventional therapy; and (3) stop conventionaltherapy and use PIF-1 alone. Patients will be continuously treated for 4weeks (using an insulin pump), with 30% dose increments, 15patients/group. Pre-therapy clinical indices, including tumor burden,will be compared to the same indices during/post PIF-1 exposure,monitoring skin for lesions and testing organ function, including PBMCability to respond to mitogen challenge.

To examine PIF-1's effectiveness in GVHD prevention with maintainedanticancer effect. Upon successful completion of the first study, BMTrecipients will be randomized into three different groups: (1)conventional therapy (control); (2) conventional prophylaxis of GVHDcombined with PIF-1; (3) PIF-1 prophylaxis alone. At transplant,patients will begin PIF-1 therapy (using an insulin pump) at 30%increments for 12 weeks followed by 3+ months observation, 15patients/group. The number of patients that develop GVHD, the degree ofthe reaction, and response to cancer will be compared between the twotest groups.

Example 5

Assess effect of PIF on PBMC isolated from patients with Crohn'sdisease. Established patients PBMC(N=20) will be isolated and culturedin the presence of PIF alone using a dose dependent design and inpresence of +/−PHA, or CD3MAb/CD28Mab, used as mitogens, using Cloningmedia, serum free. After 24 hours of exposure PBMC culture media will becollected and tested for a) cytokine release, both TH1 and TH2, usingthe Luminex 10 package b) PIF receptor expression exposing to FITC-PIFand labeled—CD14, CD4, CD8, or CD58, or CD19MAb followed by flowcytometry) c) in selective cases, mRNA will be extracted and using anAffymetrix chip global genome analysis will be carried out. Results willbe compared with PBMC similarly treated derived from normal volunteers.

Assess effect of PIF on colon biopsy of patients with Chron's disease.In parallel, to obtaining PBMC also biopsies from the same patients willbe obtained during colonoscopy. Biopsy samples will be placed in cultureto generate explants using RPMI1640 medium. Explant cultures will becarried out for 24 hours in the presence of PIF 0-200 nM. Subsequently,the media will be collected and analyzed for cytokines using the 10multiplex Luminex system (TH1 and TH2). The tissue itself will be placedin formalin and will be analyzed for cytokine content using IHC, as wellas immune cell type presence using flow cytometry and specific CDmarkers.

Generate Polyclonal Antibodies to the Synthetic PIF-1, PIF-2 and PIF-3Peptides

To generate specific antibodies against sPIF-1₁₅ (SEQ ID NO: 13)conjugation to carrier, Keyhole Limpet hemocyanin (KLH) was carried out.sPIF-1₁₅ (SEQ ID NO: 13) was conjugated to KLH either on the carboxy oramine terminus of the molecule to cover potential differences inimmunogenicity related to peptide presentation. The two peptide-carrierconjugates generated were injected into two rabbits. Within a 5-weekimmunization protocol all 4 rabbits responded by generating a high titerserum, with a titer of 1:50,000-1:150,000. The titer strength appearedto increase with the second bleeding. These rabbits may serve as along-term reservoir of serum for antibody generation AbPIF-1₁₅. Therabbits may continue to be injected with immunogens on a monthly basis,collecting sera periodically and testing for titer and affinity.Antibodies to other PIFs, including AbPIF-2₁₃ and AbPIF-3₁₈, weregenerated with the same method using KLH bound peptide in the amineterminal. Rabbits bled 8 weeks after immunization yielded 1:25,000titers for both peptides with detection of the PIF peptides to thenanomolar region. These antibodies were affinity purified using PIF-1,PIF-2 and PIF-3 bound affinity columns. The purified antibodies wereconjugated each to a separate affinity column and they will serve forisolation of PIF peptides from various biological fluids.

Monoclonal antibodies to PIF-1 were developed as well. A hybridoma cellthat produces a monoclonal antibody specific for a PIF polypeptide, andculturing the cell under conditions that permit production of themonoclonal antibody.

Such PIF antibodies may be used in assay as well as in therapeutictreatment (vaccination) of patients. For example, PIF peptide conjugatesmay be used as antigen (vaccine) to fight malaria. PIF itself, being aminimal unit might behave as a better antigen than the when its sequenceis embedded in the intact, full length circumsporozoite protein in themalaria outer cell membrane. In another example, PIF antagonist (apeptide or other chemical shown to bind to PIF receptors, and block PIFfunction) or any procedure whereby such compound is used as drug, may beuseful to treat malaria or block malaria propagation in the human body(by blocking the sites through/by which the parasite controls andparalyses the immune system and allows it to proliferate). Similarly,any humanized or horse antibodies to PIF or a procedure whereby theseare used as agents may be used for passive immunization for malaria.(assuming such antibodies must recognize the circumsporozoite protein onthe malaria parasite).

In one example, polyclonal antibodies AbPIF-1₁₅ were generated againstsPIF-1₁₅ (SEQ ID NO: 13) in rabbits (Covance Inc.). High titers 50% at1:50,000 were achieved. Serial dilutions of synthetic sPIF-1₁₅ (SEQ IDNO: 13) were plated, blocked and then washed off. PIF-1 antibody(1:5000) was added incubated and washed off. Goat anti-rabbit antibodywas added, incubated and washed off. Reaction was stopped by SDS andcounted in plate reader (Biosynthesis Inc, G Vandydriff). As shown inthe ELISA standard curve, PIF antibody detects low sPIF levels (pg). Theantibody affinity was also confirmed by using a competition analysisbetween biotin labeled and unlabeled nPIF-1₁₅ (SEQ ID NO: 1) (data notshown). Also when scrPIF-1 (SEQ ID NO: 5) was tested in the assay theantibody did not recognize it attesting to the high specificity of theantibody that was generated.

FIG. 5A demonstrates the affinity of PIF-1 IgY antibodies. Peptide astest antigen. Affi-pure IgY as the primary antibody and goat anti-Ig-Yas the secondary antibody. A fixed amount of antigen (5 ug/ml) andserial dilution of IgY.

FIG. 5B demonstrates the specificity of PIF-1 polyclonal antibody. At4.5 ug/ml pAb coating concentration, PIF-1₁₅ was detectable at 10-30 μMin a dose response curve with an IC₅₀ of 500-700 pm, and linearly up to30 nM. scrPIF-1 did not compete with biotinylated peptide, as thenative. No binding appears to occur on uncoated plates, yielding a goodbackground. Results demonstrate that PIF-1 polyclonal antibody appearsto avoid false positives and negatives.

In one non-limiting embodiment, a PIF-based pregnancy diagnosisutlilizing an ELISA or yes/no stick in the form of a kit is provided.The components of the PIF ELISA kit may include, for example,HRP-Avidin, PIF-Biotin and anti-PIF-1₁₅ antibody. In the absence of PIFin the test sample, HRP enzyme would bind to the antibody through thePIF-biotin complex, generating a maximum color. In the presence of PIFin the test sample, PIF binds to the PIF antibody and prevents the HRPenzyme complex from binding, generating a minimum color.

Isolation and Identification of PIF Like Proteins in Human Placenta andOther Fetal Tissues

Using affinity purified PIF IgG 1, 2 and 3 and Igy PIF-1, PIFs wereidentified in human term placenta using Western blot. Human placenta wasthe test antigen. Lanes 1 and 3 were loaded with 50 ug of antigen perlane and lanes 2, 4 and 5 were loaded with 100 ug of antigen per lane.Lanes 1 and 2 were incuabeted with affi-pure anti-PIF-1 igY in a 1:50dilution, goat anti-IgY-HRP in a 1:1000 dilution. Lane 3 was incubatedwith anti-PIF-1 antibody in 1:200 dilution. Lane 4 was incubated withanti-PIF-2 antibody in 1:50 dilution. Lane 5 was incubated withanti-PIF-3 antibody in 1:50 dilution and goat/anti-rabbit-HRP in adilution of 1:1000. Results of western blot are shown in FIG. 6.

Expression of PIF-1 in human pregnancy tissues was examined usingaffinity purified IgG using immunohistochemistry methods. Intensetrophoblastic expression was found in first and second trimesterplacenta while expression was low at term. With respect to the 14-18weeks fetus using a tissue array (60 samples, covering practically allorgans). The highest expression was in the spleen and liver, with lesserin the adrenal, stomach and small bowel with no detectable expression inthe esophagus and several other organs. The presence of PIF was alsomeasured in the adrenal tissue, stomach, small bowel, thyroid and otherorgans (not shown). Non relevant IgG was used as controls.

Overall this indicates that PIF-1 is expressed in the human placenta andfetus across gestation where it declines at term to facilitate theprocess of delivery by lowering maternal tolerance for the fetus. Withrespect for the fetus highest expression are found in hemopoietic organswhere immune reaction is expected to be the highest.

In another example, PIF-1 associated proteins were identified in humanplacental tissue. Term human placental homogenates were passed throughan affinity column of PIF-1 antibody. The mass spectrometry profilefollowing elution by PIF-1 antibody affinity column. Various PIF-1associated proteins were identified and sequenced following affinitychromatography, including (NM_(—)000039) apolipoprotein A-I precursor[Homo sapiens], electron-transferring-flavoprotein dehydrogenase,(BC017165) similar to triosephosphate isomerase 1 [Homo sapiens],(NM_(—)052925) leukocyte receptor cluster (LRC) member [Homo sapiens],(NM_(—)018141) mitochondrial ribosomal protein S10; mitochondrial 28Sribosomal protein S10 [Homo sapiens], (NM_(—)000518) beta globin [Homosapiens], (BC012292) heat shock 27 kDa protein 1, stress responsive,estrogen regulated [Homo sapiens] P04792.40, Estradiol beta 1dehydrogenase 1 P14061.13, Fetal Beta MHC binding factor Q 14297.01,microtubule-associated protein 1A (proliferation-related protein p80P78559.40). Some of those proteins were not previously described in theplacenta. The proteins sequenced appear to show roles in immunefunction, cytoskeleton, enzyme function, and protein synthesis and cellproliferation. None of the sequenced proteins have sequence homologywith PIF-1, therefore it likely reflects, in some cases, that PIF isattached to these proteins reflecting a protein-protein interactionrelated to the peptides function.

Demonstration that PIF is Present in the Placenta of Sheep

Placental tissues were collected from a mid-gestation sheep fetus. Theplacenta was embedded in paraffin and slides were prepared.Representative slides exposed to the 1/100 dilution of rabbit AbPIF-1₁₅antibody. Compared with the non immunized serum, AbPIF-1₁₅ antibodyintensely stained the placenta, as shown in FIG. 8. The DAKO Chemmatesystem on the autostainer with DAB as the substrate was used. Moreover,the binding was highly specific since no adjacent maternal tissuesappeared to be stained by the antibody. As such, AbPIF-1₁₅ antibody aswell as AbPIF-2₁₃ and AbPIF-3₁₈ could be useful in identifying presenceof PIF in pregnant tissues. Gestational changes: Visual analysis wascarried out on day 50 (n=4), day 80 (n=7), day 100 (n=7), day 128 (n=4)and day 135 (n=8) 9Term=145 days). Based on visual assessment of percentstaining, placental levels were highest at day 50 and then declined today 80 after which they remained constant. This pattern reflected theobservations that were made with the human placenta.

In terms of localization, PIF-1 is localized to the ovine maternal-fetalinterface which is comprised of fetal trophoblast and maternalepithelium. Interestingly, PIF-1 is localized to the binucleatetrophoblast cells. These are non-proliferative migratory cells whichfuse with the maternal epithelium to form a hybrid maternal-fetalsyncytium. On many of the slides there appears to be epithelialstaining. Some of this may come from the fetal contribution to thislayer. Later in gestation, the staining becomes more restricted to thebinucleate trophoblastic cell population.

PIF-1 expression decreases at term of growth restricted fetuses. Acomparison of the placental growth restricted group (n=14) to thecontrols (n=14) at day 80 (placental growth period) gave no significantdifferences. In late gestation, there is evidence of a decrease in PIF1in the growth restricted group compared with controls.

Preliminary Study: PIF-1 Antibody and Scrambled PIF-1 IntravenousAdministration is Non Toxic

The contraceptive effect of either 150 ug of PIF-1scr in DMSO or 10 ugaffinity purified PIF-1 (10 animals per group) or 20% DMSO solution(used as controls) was tested using single daily intravenous injections(Dr Alan Hoberman, Argus, Inc). Female mice were placed with male miceon the 3rd day afternoon of the expected estrus. Mating was confirmed bythe presence of sperm in the vagina or a copulatory plug the nextmorning. Subsequently, were injected for 5 days one injection/day. Micewere sacrificed at day 12 of presumed gestation and Caesarean-sectioned.Corpora lutea, implantation sites and number of live and dead embryoswere recorded. 2/10 mice in the PIF-1a and PIF-1 antibody group did notget pregnant, while in the control group all mice were pregnant. Theeffect was all or none since no toxic or teratogenic effects were notedin the mice that conceived.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

1. An antibody that binds specifically to PIF polypeptide comprising theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12 or afragment thereof having at least 8 amino acids.
 2. The antibody of claim1, comprising AbPIF-1₁₅, AbPIF-2₁₃ or AbPIF-3₁₈.
 3. The antibody ofclaim 1, wherein the antibody comprises a monoclonal or polyclonalantibody.
 4. A composition for detecting the presence of PIF peptide ina fluid, the composition comprising: PIF antibodies; and the fluid. 5.The composition of claim 4, wherein the fluid is selected from the groupconsisting of serum, blood, urine, milk, saliva, embryo culture media,gestational tissues, fetal tissue and combinations thereof.
 6. Thecomposition of claim 4, wherein the antibodies are labeled.
 7. Thecomposition of claim 4, wherein the antibodies are chemically bonded toa substrate.
 8. The composition of claim 4, wherein the antibodiescomprise a monoclonal or polyclonal antibody.
 9. A method of assayingPIF in a sample comprising, obtaining a sample from a subject, mixingthe sample with a detecting antibody and measuring the concentration ofPIF in the sample.
 10. The method of claim 9, wherein the antibodyassay, is an ELISA.
 11. The method of claim 10, wherein the assay is asandwich ELISA.